The broad objective of this proposal is to understand the interactions of transcriptional regulatory proteins with specific DNA sequence elements and the mechanisms by which these interactions modulate transcription frequency of genes in animal cells. Regulation at this level is critical in determining the developmental fate and function of cells in both normal and disease states. The transcriptional regulation of heat shock genes of Drosophila is to be used as a well-defined and highly-inducible model system. The binding of the heat shock regulatory factor, HSF, to the regulatory elements, HSEs, of heat shock genes is postulated to act catalytically to increase the rate of transcription of adjacent genes. The first section of the grant focuses on binding of HSF to the HSEs, while the second focuses on a potential target for the catalytic action of HSF. The aims of Section 1 are to investigate the properties and significance of a novel type of protein/DNA interaction between HSF and HSEs. The remarkable flexibility in binding of HSF to different arrangements of a repeated 5 bp recognition unit of HSEs can be explained by recent findings that HSF from induced cells is in the form of a protein trimer. The trimeric nature of HSF has evoked a number of predictions about the structure HSF/HSE complexes that this proposal will test by in vitro binding and interference assays. The biological significance of the strong cooperativity of HSF binding to HSEs observed in vitro will be evaluated by measuring transcription of native and modified heat shock genes using run- on assays of nuclei isolated from transgenic flies. This aims of Section 2 of the proposal are to investigate the formation and release of the paused (arrested) RNA polymerase II that exists on the 5' end of the hsp70 and hsp26 heat shock genes in uninduced cells and on the 5' end of at least some constutively expressed Drosophila genes. The main focus is on heat shock genes where release of polymerase beyond the pause site is rate-limiting in uninduced cells, and thus, is a potential target for the catalytic action of HSF during the heat shock response. The features of promoters that generate paused RNA polymerases will be identified by run-on assays performed with nuclei from uninduced transgenic fly lines containing altered hsp70 genes. The sites of pausing in vivo on native and altered heat shock genes will be examined at nucleotide resolution to determine whether the pause site is dictated by specific sequence elements or other features of the promoter. The release of paused polymerase in nuclei by addition of purified HSF and other nuclear fractions will be examined. Attempts will be made to establish an in vitro system that places a paused polymerase on an hsp70 gene template so that the mechanisms by which it is generated and released can be investigated.